Recent advances in the biodegradation of polyethylene terephthalate with cutinase-like enzymes.

Polyethylene terephthalate (PET) is a synthetic polymer in the polyester family. It is widely found in objects used daily, including packaging materials (such as bottles and containers), textiles (such as fibers), and even in the automotive and electronics industries. PET is known for its excellent mechanical properties, chemical resistance, and transparency. However, these features (e.g., high hydrophobicity and high molecular weight) also make PET highly resistant to degradation by wild-type microorganisms or physicochemical methods in nature, contributing to the accumulation of plastic waste in the environment. Therefore, accelerated PET recycling is becoming increasingly urgent to address the global environmental problem caused by plastic wastes and prevent plastic pollution. In addition to traditional physical cycling (e.g., pyrolysis, gasification) and chemical cycling (e.g., chemical depolymerization), biodegradation can be used, which involves breaking down organic materials into simpler compounds by microorganisms or PET-degrading enzymes. Lipases and cutinases are the two classes of enzymes that have been studied extensively for this purpose. Biodegradation of PET is an attractive approach for managing PET waste, as it can help reduce environmental pollution and promote a circular economy. During the past few years, great advances have been accomplished in PET biodegradation. In this review, current knowledge on cutinase-like PET hydrolases (such as TfCut2, Cut190, HiC, and LCC) was described in detail, including the structures, ligand-protein interactions, and rational protein engineering for improved PET-degrading performance. In particular, applications of the engineered catalysts were highlighted, such as improving the PET hydrolytic activity by constructing fusion proteins. The review is expected to provide novel insights for the biodegradation of complex polymers.

A tomato chloroplast-targeted DnaJ protein, SlDnaJ20 maintains the stability of photosystem I/II under chilling stress.

DnaJ proteins are key molecular chaperones that act as a part of the stress response to stabilize plant proteins, thereby maintaining protein homeostasis under stressful conditions. Herein we used transgenic plants to explore the role of the tomato (Solanum lycopersicum) SlDnaJ20 chloroplast DnaJ protein in to the resistance of these proteins to cold. When chilled, transgenic plants exhibited superior cold resistance, with reduced growth inhibition and cellular damage and increased fresh mass and chlorophyll content relative to control. These transgenic plants further exhibited increased Fv/Fm, P700 oxidation, φRo, and δRo relative to control plants under chilling conditions. Under these same cold conditions, these transgenic plants also exhibited higher levels of core proteins in the photosystem I (PSI) and II (PSII) complexes (PsaA and PsaB; D1 and D2) relative to control wild-type plants. Together these results suggested that the overexpression of SlDnaJ20 is sufficient to maintain PSI and PSII complex stability and to alleviate associated photoinhibition of these complexes, thereby increasing transgenic plant resistance to cold stress.

Potential molecular targets of nonstructural proteins for the development of antiviral drugs against SARS-CoV-2 infection.

Outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have produced high pathogenicity and mortality rates in human populations. However, to meet the increasing demand for treatment of these pathogenic coronaviruses, accelerating novel antiviral drug development as much as possible has become a public concern. Target-based drug development may be a promising approach to achieve this goal. In this review, the relevant features of potential molecular targets in human coronaviruses (HCoVs) are highlighted, including the viral protease, RNA-dependent RNA polymerase, and methyltransferases. Additionally, recent advances in the development of antivirals based on these targets are summarized. This review is expected to provide new insights and potential strategies for the development of novel antiviral drugs to treat SARS-CoV-2 infection.

Thymosin-ß12 characteristics and function in Urechis unicinctus.

ß-thymosin family comprise a series of heat-stable multifunctional polypeptides involved in actin regulation, anti-inflammation, wound healing, cell migration, angiogenesis, cardiac protection, antimicrobial processes and antiviral immunity. The roles of Tß12 (thymosin-ß12) in marine invertebrates is still largely unknown, especially in terms of antibacterial immunity. In this study, we cloned the Tß12 gene with an ORF of 126 bp coding 41 amino acids from Urechis unicinctus. Tissue distribution analysis by qRT-PCR used TBP as reference gene showed that Tß12 was widely expressed in all tissues, and the transcript levels were the highest in the body wall, followed by the coelomic fluid, and the lowest in the intestines and anal sacs. After LPS (lipopolysaccharides) injection, Tß12 expression in the body was first elevated significantly at 3 h (p < 0.05), indicating that the body wall was the first defense line of the innate immune system; in the coelomic fluid, the Tß12 mRNA levels increased after LPS injection, with a significant increase occurring at 6 h, showing that coelomic fluid functioned as the second defense line of the innate immune system. In the midgut and anal sacs, a significant increase in the Tß12 level occurred at 24 h, suggesting that the midgut and anal sacs may act as accessory organs for the innate immune system. Moreover, U. unicinctus Tß12 recombinants can effectively inhibit the growth of both gram-negative and gram-positive bacteria. These results indicate that U. unicinctus Tß12 plays important roles in innate antibacterial immune responses, which can deepen our understanding of Tß12 in marine invertebrates.

Strategy for the Biosynthesis of Short Oligopeptides: Green and Sustainable Chemistry.

Short oligopeptides are some of the most promising and functionally important amide bond-containing components, with widespread applications. Biosynthesis of these oligopeptides may potentially become the ultimate strategy because it has better cost efficiency and environmental-friendliness than conventional solid phase peptide synthesis and chemo-enzymatic synthesis. To successfully apply this strategy for the biosynthesis of structurally diverse amide bond-containing components, the identification and selection of specific biocatalysts is extremely important. Given that perspective, this review focuses on the current knowledge about the typical enzymes that might be potentially used for the synthesis of short oligopeptides. Moreover, novel enzymatic methods of producing desired peptides via metabolic engineering are highlighted. It is believed that this review will be helpful for technological innovation in the production of desired peptides.

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum.

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384 × 106 Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30 °C and 180 rpm: carbon source, 50 g/L maltose; initial pH 7; and 8 g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3 L. The fermentation broth contained 303 g/L maltose, which produced 122.34 g/L pullulan with an average productivity of 1.0195 g/L/h and 82.32 g/L dry biomass within 120 h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251 g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.

A systematic optimization of styrene biosynthesis in Escherichia coli BL21(DE3).

BACKGROUND: Styrene is a versatile commodity petrochemical used as a monomer building-block for the synthesis of many useful polymers. Although achievements have been made on styrene biosynthesis in microorganisms, several bottleneck problems limit factors for further improvement in styrene production. RESULTS: A two-step styrene biosynthesis pathway was developed and introduced into Escherichia coli BL21(DE3). Systematic optimization of styrene biosynthesis, such as enzyme screening, codon and plasmid optimization, metabolic flow balance, and in situ fermentation was performed. Candidate isoenzymes of the rate-limiting enzyme phenylalanine ammonia lyase (PAL) were screened from Arabidopsis thaliana (AtPAL2), Fagopyrum tataricum (FtPAL), Petroselinum crispum (PcPAL), and Artemisia annua (AaPAL). After codon optimization, AtPAL2 was found to be the most effective one, and the engineered strain was able to produce 55 mg/L styrene. Subsequently, plasmid optimization was performed, which improved styrene production to 103 mg/L. In addition, two upstream shikimate pathway genes, aroF and pheA, were overexpressed in the engineered strain, which resulted in styrene production of 210 mg/L. Subsequently, combined overexpression of tktA and ppsA increased styrene production to 275 mg/L. Finally, in situ product removal was used to ease the burden of end-product toxicity. By using isopropyl myristate as a solvent, styrene production reached a final titer of 350 mg/L after 48 h of shake-flask fermentation, representing a 636% improvement, which compared with that achieved in the original strain. CONCLUSIONS: This present study achieved the highest titer of de novo production of styrene in E. coli at shake-flask fermentation level. These results obtained provided new insights for the development of microbial production of styrene in a sustainable and environment friendly manner.

Pullulan production from synthetic medium by a new mutant of Aureobasidium pullulans.

Pullulan with different molecular-weight could be applied in various fields. A UV-induced mutagenesis Aureobasidium pullulans UVMU6-1 was obtained from the strain A. pullulans CGMCC3.933 for the production of low-molecular-weight pullulan. First, the obtained polysaccharide from A. pullulans UVMU6-1 was purified and identified to be pullulan with thin-layer chromatography, Fourier transform infrared, and nuclear magnetic resonance. Then, culture medium and conditions for this strain were optimized by flask fermentation. Based on the optimized medium and culture conditions (pH 4, addition of 4 g/L Tween 80 for 96 hr of cultivation), continuously fermentation was performed. The highest pullulan production and dry biomass was 109 and 125 g/L after fermentation for 114 hr, respectively. The average productivity was about 1 g/L/hr, which was intensively higher than the previous reported. This study would lay foundations for the industrial production of pullulan.

An integrative process of bioconversion of oil palm empty fruit bunch fiber to ethanol with on-site cellulase production.

The aim of this study was to efficiently convert oil palm empty fruit bunch fiber (OPEFB), one of the most commonly generated lingo-wastes in Southeast Asia, into both cellulase and bioethanol. The unprocessed cellulase crude (37.29%) produced under solid-state fermentation using OPEFB as substrate showed a better reducing sugar yield using filter paper than the commercial enzyme blend (34.61%). Organosolv pretreatment method could efficiently reduce hemicellulose (24.3-18.6%) and lignin (35.2-22.1%) content and increase cellulose content (40.5-59.3%) from OPEFB. Enzymatic hydrolysis of pretreated OPEFB using the crude cellulase with 20% solid content, enzyme loading of 15 FPU/g OPEFB at 50 °C, and pH 5.5 resulted in a OPEFB hydrolysate containing 36.01 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 17.64 g/L ethanol with 0.49 g/g yield from glucose and 0.088 g/g yield from OPEFB at 8 h using Saccharomyces cerevisiae.